整合人乳铁蛋白cDNA山羊胎儿成纤维细胞系的建立

通过转基因动物乳腺表达系统生产重组药用蛋白比微生物发酵系统和动物细胞培养系统具有很多优势；同时，体细胞核移植技术为制备动物乳腺生物反应器开辟了一条新的途径。本实验旨在建立稳定整合人乳铁蛋白cDNA的山羊胎儿成纤维细胞系，为体细胞核移植法制备转基因动物提供可靠的供体细胞。本研究采用RT-PCR获得人乳铁蛋白cDNA，通过Long and Acute PCR扩增山羊β-酪蛋白5’ 端6.5 kb的调控序列，以pEGFP-C1为骨架构建乳腺特异性表达载体 p6.5hLF-EGFP。脂质体介导法转染山羊胎儿成纤维细胞，G418抗性筛选3-4周后，经PCR扩增和报告基因EGFP表达检测，得到稳定整合外源基因的转基因供体细胞系，为制备高效表达人乳铁蛋白的转基因山羊乳腺生物反应器提高可靠的核移植供体细胞。

Establishment of Human Lactoferrin cDNA Transgenic Caprine Fetal Fibroblast Cell Lines

The production of recombinant human pharmaceuticals in the milk of transgenic farm animals has many advantages when compared with microbial bioreactors or animal cell bioreactors. At the same time, somatic cell nuclear transfer (SCNT) has provided an alternative avenue to the production of transgenic animals. The purpose of this study was to establish human lactoferrin cDNA transgenic donor cell lines and to prepare competent donor cells for the production of transgenic animals by SCNT. Human lactoferrin cDNA was obtained by RT-PCR while 6.5 kb caprine β-casein regulatory elements were amplified by Long and Acute PCR. Subsequently the two fragments were inserted into the corresponding site of the plasmid pEGFP-C1, resulting in the mammary specific expression vector p6.5hLF-EGFP. Then the caprine fetal fibroblast cells were transfected with p6.5hLF-EGFP by LipofectamineTM-2000 and selected by G418 for 3 to 4 weeks. The G418 resistant transfectants were identified by PCR and EGFP detection. The results indicated that the transgene was stably integrated into the open region of the chromatin of G418 resistant fibroblast cells. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.